Tuesday, August 25, 2009

Plasmid DNA Extraction Using Alkaline Lysis Method

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.

  • Luria Bertani (LB) broth bacteria culture medium: 1% Tryptone, 0.5% yeast extract, 200 mM NaCl. Sterilize by autoclaving in suitable aliquots. In order to ensure retention of the plasmid, media should be supplemented with the appropriate antibiotic(s).
  • 1.5 mL Microfuge tubes.
  • Sterile tubes: Must have a volume of at least 10 mL to ensure good aeration.
  • Lysis solution: 200 mM NaOH, 1% SDS. Store at room temperature.
  • Resuspension solution: 50 mM glucose, 50 mM Tris-HCl, pH 8.0, 10 mM ethylene diamine tetraacetic acid (EDTA). Keep at 4oC to prevent growth of contaminants.
  • Potassium acetate (neutralizing solution): 3 M potassium/5 M acetate. For 100 mL, take 29.4 g of potassium acetate, add water to 88.5 mL, and 11.5 mL of glacial acetic acid. Store at room temperature.
  • TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
  • Isopropanol.
  • 70% Ethanol.

  • Take a number of separate sterile tubes and place 2 mL of L-broth into them. Inoculate from individual bacterial 37°C overnight with shaking.
  • Transfer each culture to a labeled 1.5-mL Eppendorf tube, and centrifuge for 30 s at high speed in the microfuge.

    Here, a tight creamy pellet may be seen.

  • Decant the supernatant and place tubes in a rack vertically for 5-10 s. Remove any of the remaining liquid by aspiration with a fine Pasteur pipet.
  • Add 100 microliters of resuspension solution into each tube, close the lids, and resuspend the bacteria in each tube by shaking or vortexing to dissociate the bacterial pellet.
  • To each tube add 200 microliters of lysis solution and mix by inverting the tube several times.

    The solution should quickly turn transparent and become more viscous indicating bacterial
    lysis has taken place.

  • Allow at least 2-3 min for lysis to take place and leave the tubes to stand for 60 s before opening. This will allow the liquid to return to the bottom of the tube.
  • To each tube add 150 microliters of neutralizing solution and invert the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
  • Centrifuge the tubes for 2-5 min at full speed in a microfuge.
  • Place new sterile tubes into a rack, label them, and add 250 ~ of isopropanol to each tube.
  • Remove the tubes from the microfuge, being careful not to disturb the precipitate.
  • Remove the supernatant with a 1-mL pipet, avoiding the white precipitate as much as possible.

    Some of the precipitate may float, so it is critical to use a pipet and disposable tips to
    recover the supernatant rather than pouring it.

  • Transfer the liquid phase into the new set of labeled tubes containing the isopropanol.
  • Vortex the tubes for 5-10 s and centrifuge the tubes in the microfuge for 30 s at high speed. The plasmid DNA precipitates as a white pellet.
  • Decant the supernatant and wash the pellets by adding 750 mL of 70% ethanol, vortex briefly, and centrifuge at high speed for 30 s.
  • Decant the ethanol, and centrifuge again for 10 s to collect the remaining ethanol at the bottom of the tubes. Carefully aspirate the remaining ethanol and leave the tubes to air dry on the bench for 5 min.
  • Dispense 50 microliter of TE into each tube, and resuspend the pellet

    It is not advisable to vortex, as this may lead to DNA shearing. The sample is best left for
    3-5 min with occasional finger flicking of the tube.

  • Take 10 microliters of the resuspended pellet and analyze by agarose gel electrophoresis (see on my posting entitled Agarose Gel Electrophoresis of Nucleic Acids).

Actually, you can use another method to extract plasmid DNA instead of Alkaline Lysis. I will try to describe it on the next post. Hopefully, this method will be useful for your lab work.

Reference: Number 18 on References